Genome-Wide Location Analyses of N6-Methyladenosine Modifications (m6A-Seq)

Benoit Molinie; Cosmas C Giallourakis

doi:10.1007/978-1-4939-6807-7_4

Genome-Wide Location Analyses of N6-Methyladenosine Modifications (m⁶A-Seq)

Methods Mol Biol. 2017:1562:45-53. doi: 10.1007/978-1-4939-6807-7_4.

Authors

Benoit Molinie¹, Cosmas C Giallourakis²

Affiliations

¹ Gastrointestinal Unit, 55 Fruit Street, Jackson 828, Boston, MA, 02114, USA. molinie.benoit@gmail.com.
² Gastrointestinal Unit, Massachusetts General Hospital, Harvard Medical School, 55 Fruit Street, Jackson 828, Boston, MA, 02114, USA. cgiallourakis@mgh.harvard.edu.

Abstract

N⁶-methyladenosine-sequencing (m⁶A-seq) is a critical tool to obtain an unbiased genome-wide picture of m⁶A sites of modification at high resolution. It allows the study of the impact of various perturbations on m⁶A modification distribution and the study of m⁶A functions. Herein, we describe the m⁶A-seq protocol, which entails RNA immunoprecipitation (RIP) performed on fragmented poly(A) RNA utilizing anti-m⁶A antibodies. The captured/enriched m⁶A positive RNA fragments are subsequently sequenced by RNA-seq in parallel with background control non-immunoprecipitated input RNA fragments. Analyses reveal peaks of m⁶A enrichment containing sites of modifications analogous to chromatin modification immunoprecipitation experiments.

Keywords: Epitranscriptome; Genome-wide; METTL14; METTL3; N6-Methyladenosine; m6A-Seq.

MeSH terms

Adenosine / analogs & derivatives*
Computational Biology / methods
Gene Expression Profiling
Genome*
Methylation
RNA* / chemistry
RNA* / isolation & purification
RNA* / metabolism
RNA, Messenger / chemistry
RNA, Messenger / isolation & purification
RNA, Messenger / metabolism
Sequence Analysis, RNA*
Transcriptome
Workflow

Substances

RNA, Messenger
RNA
N-methyladenosine
Adenosine

Grants and funding

P30 DK043351/DK/NIDDK NIH HHS/United States